Functional, endogenously expressed corticotropin-releasing factor receptor type 1 (CRF1) and CRF1 receptor mRNA...

Corticotropin-releasing factor (CRF) is a hypothalamic 41-amino acid peptide which stimulates corticotropin (ACTH) release from the anterior pituitary and is also involved in the body response to stress. CRF1 receptors represent a potential target for novel antidepressant/anxiolytic drugs. The aim of the present study was to search for a human cell line expressing native, functional CRF1 receptors as a starting material for screening purposes. We identified CRF1 receptors functionally coupled to cAMP formation in human neuroblastoma SH-SY5Y cells. CRF induced concentration-dependent increases in cAMP accumulation in SH-SY5Y cells (maximal increase 6.9 +/- 0.9 fold over basal values, n = 14). This effect was mimicked by related peptides with similar potencies: (mean pEC50 value) human/rat CRF (8.63), rat urocortin (9.32), sauvagine (8.97), urotensin I (8.93), ovine CRF (8.81). The efficacies of these agonists were nearly the same, with the exception of ovine CRF which was slightly less efficacious (75% the Emax of CRF). The responses to CRF were competitively antagonised by the following peptide fragments (mean pKB value): alpha-helical-CRF (9-41) (7.54), [D-Phe12,Nle21,38,C alpha MeLeu37]CRF (12-41) (8.36) and [D-Tyr12]astressin (9.49) and by the selective, non-peptidic CRF1 receptor antagonists, CP-154,526 (7.76) and antalarmin (9.19). Estimation of receptor density by [125I]Tyr0-ovine CRF saturation binding yielded a modest number of binding sites (Bmax 12 fmol/mg protein, KD 0.2 nM). Analysis of mRNA by reverse transcription-polymerase chain reaction clearly revealed the presence of mRNA for CRF1 receptors in SH-SY5Y cells. A slight signal for CRF2 receptor mRNA was also observed. We conclude that neuroblastoma SH-SY5Y cells are endowed with native CRF1 receptors positively coupled to cAMP formation. They therefore constitute a useful functional model for the search of CRF1 selective compounds with potential anxiolytic/antidepressant activity.